Team:UIUC Illinois/Results
Results Overview
In order to gauge Roundown's response to both pure glyphosate and commercial formulations of glyphosate-based herbicides, we measured its growth over time relative to control plasmid carrying cell line. Detailed protocols for these experiments can be found in the Protocols section. We attempted to induce individual expression of each gene using the promoters pLac and pTet but ran into issues with inhibiting the pTet promoted gene. This meant that the gene glpA, which is associated with increased glyphosate tolerance, being constitutively expressed. In experiments where IPTG is added, the expression of glpB is induced and both genes are operational.
Growth in Roundup
The following Figure 1 shows the growth of Rounddown relative to the control when both genes were induced when grown in the commercial herbicide Roundup. Rounddown showed improved growth relative to the control. At 3 mM the control displayed a much longer lag phase than Rounddown and at concentrations greater than 3 it was unable to grow at all. Rounddown reached similar OD600 values for growth conditions up to 4 mM Roundup. In 5 mM Roundup its peak OD was reduced but it still grew better than the control at this condition.
Growth in Glyphosate
The next graph, Figure 4 shows the relative growth of Rounddown when grown in pure glyphosate. While increasing the concentration of glyphosate had limited effect on the peak OD600 reached by the cells, it did extend the lag phase of Rounddown and the control for all increases in concentration. Rounddown was able to reach a similar peak OD for concentrations up to 13 mM over the time period the samples were measured. At 13 mM Rounddown reached a peak OD of around 1.2, while the control only reached an OD of 0.4. It is also interesting to note that the peak OD reached by Rounddown was greater for samples containing glyphosate than the samples grown in just LB. This may be due to utilization of glyphosate as a phosphate source.
glpA Testing
glpA action is believed to be associated with the transport of glyphosate across the cell membrane due to it's homology with native E. coli phosphotransferases. In order to gauge this gene's impact on the ability of the cell to survive the presence of glyphosate, it was tested in both pure glyphosate as well as a commercial formulation of the herbicide, Roundup. Growth curve data was used to determine if glpA confers a survival advantage the cells compared to other E. coli without the gene. The promoter for this gene was pTet but as the cell line used lacked tetR, glpA was constitutively expressed.
When grown in pure glyphosate, the construct Rounddown showed very little difference in growth compared to the control when only glpA was expressed. This can be seen in the following figure, which displays the growth curves obtained when Rounddown and the control were grown in pure glyphosate with no IPTG. Peak OD600 decreased for both cell types with each increase in concentration.
When grown in Roundup, a commercial formulation containing surfactants in addition to glyphosate, the presence of glpA had a profound effect. As shown in the following figure, the presence of glpA allowed cells to survive at concentrations up to 4 mM in Roundup, while cells that did not contain the gene were unable to grow past 2 mM concentrations of Roundup. The control showed a very long lag phase when grown in 3 mM, and growth was just starting to increase as the end of the measurement period occurred. The peak OD600s for Rounddown at concentrations of 2 mM, 3 mM, and 4 mM were all very similar, though they were moderately lower than Rounddown grown in the absence of herbicide.
While it is unexpected that there is no difference in growth seen between Rounddown and the control in pure glyphosate, there is a very clear advantage for Rounddown when grown in Roundup. Roundup in general is more toxic, as growth inhibition is seen at much lower concentrations of glyphosate than when grown in a solution containing a pure form of the molecule. This would indicate that the surfactant or some part of the formulation is negatively impacting growth and that glpA helps the cell to survive in the presence of these factors as well as in the presence of glyphosate. In terms of applications in bioremediation, it is preferable that the gene provides an advantage for a formulation of the herbicide, since all large scale herbicidal applications use formulated products.
glpB Testing
glpB was used as part of a two part system with the goal of increasing cell survivability and degradation of glyphosate. glpB is associated with the breakdown of glyphosate through the glyphosate oxidoreductase pathway, in which the glyphosate molecule is broken at the C-N bond to form aminomethylphosphonic acid (AMPA) and glyoxylate. In our system, glpB was regulated by pLac, and in experiments expression was induced by adding IPTG.
Compared to when only glpA was expressed, the induction of glpB seemed to improve growth in pure glyphosate. The following figure shows the growth in pure glyphosate when IPTG was added to compared to when it wasn't present. In the presence of IPTG, Rounddown was able to grow to a higher peak OD than when there was no IPTG added. This could be potentially be because the cells are able to utilize the glyphosate as a phosphate source. In the presence of IPTG the Rounddown also showed a much shorter lag phase, reaching peak OD at a much faster rate.
The main effect of glpB in a pure glyphosate system it to decrease the length of the lag phase. A slightly different effect is seen in a system utilizing Roundup. When IPTG is added to cells growing in Roundup, the peak OD reached by the cells is very similar to that reached when no Roundup is present. This is shown in the following figure. This increase in peak OD is indicative of increased tolerance towards the glyphosate and surfactant mixture than when glpA alone is expressed.
Testing Degradation
To test the efficiency of E. coli degrading glyphosate, we chose NMR, a method doesn’t heavily rely on devices, i.e., column and testing environment, i.e., mobile phases. NMR result for preliminary testing shows that E. coli doesn't degrade glyphosate as expected because glyphosate concentration does not decreases significantly after a culture cycle, i.e., 3 days. One of the hypotheses is that glyphosate is not transferred into the pathway fast enough to show a decrease of glyphosate concentration in media.
To test the efficiency of Rounddown degrading glyphosate, we chose NMR, since it’s a fairly simple method that doesn’t require lots of materials. NMR results for preliminary testing shows that E. coli doesn’t degrade glyphosate as expected since the glyphosate concentration does not decrease significantly after a culture cycle, which is approximately 2 days for Rounddown.
We have several hypotheses for why this didn’t work. Our first hypothesis is that we didn’t allow the samples to grow for enough time, since though bacterial growth reaches saturation after 2 days, it may take longer for Rounddown to begin degrading glyphosate. Another hypothesis is that glyphosate’s spectra potentially overlaps with the spectra of glyphosate’s natural degradation product, aminomethylphosphonic acid (AMPA). This would lead to an inability to test for differences in concentrations since if AMPA was being secreted, it would simply replace the glyphosate that was degraded when quantified using NMR. Due to a lack of time to test the various controls required to successfully quantify glyphosate at the present, we plan to carry forward with gathering more quantification data in the future.
Future Goals
Additional testing and direct detection of glyphosate would be useful in determining the efficacy of the two genes, in particular the activity of glpB in biodegradation of glyphosate. Additionally, transferring the plasmids into a bacterium native to the soil, such as Bacillus subtilis, would be useful as such a bacterium would have greater potential for use in bioremediation. Testing the strain's ability to grow in more realistic field conditions would also be an important step towards the genes use in practical applications.
